Bottom Line:
ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs.The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment.In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

ABSTRACTIn this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

f1: Effect of ICA treatment on the viability and morphology of human ESCC cells.(A) EC109 cells were treated with increasing concentrations of ICA (20, 40, or 80 μM) and assessed at different time points (12, 24, and 36 h). The cell viability is expressed as OD values. (B) TE1 cells were treated with increasing concentrations of ICA (20, 40, or 80 μM) and assessed at different time points (12, 24, and 36 h). The cell viability is expressed as OD values. The morphology of both of the ESCC lines was observed under an inverted phase-contrast microscope after the cells were treated for 24 h, and images were obtained. Significant cell shrinkage and a decreased cellular attachment rate were observed in the ICA treatment groups. All of the results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the 20 μM ICA-treated group; cP < 0.05 vs. the 40 μM ICA-treated group.

Mentions:
To investigate whether ICA displays antitumor activity in EC109, TE1, and HET-1A cells, the CCK-8 assay was used to evaluate the effects of ICA on different cells, and the data are shown in Fig. 1. Treatment of EC109 and TE1 cells for 12, 24, or 36 h with 20, 40, or 80 μM ICA inhibited cell viability in a dose- and time-dependent manner. The 50% inhibitory concentrations (IC50s) of ICA at 12, 24, and 36 h were approximately 106.13 μM, 73.65 μM, and 38.59 μM in EC109 cells and 115.29 μM, 76.77 μM, and 42.21 μM in TE1 cells, respectively. Microscopic images (EC109 in Fig. 1A and TE1 in Fig. 1B) indicated that compared with the control treatment ICA treatment resulted in significant cell shrinkage (based on the bar and cellular gap) and reduced the rate of cellular attachment (based on the cell numbers). However, ICA at the same concentrations did not affect the survival of human primary esophageal epithelial HET-1A cell (Supplementary Fig. 2). The results suggest that icariin has promising anti-ESCC activity with low cytotoxic effect on normal esophageal epithelial cell.

f1: Effect of ICA treatment on the viability and morphology of human ESCC cells.(A) EC109 cells were treated with increasing concentrations of ICA (20, 40, or 80 μM) and assessed at different time points (12, 24, and 36 h). The cell viability is expressed as OD values. (B) TE1 cells were treated with increasing concentrations of ICA (20, 40, or 80 μM) and assessed at different time points (12, 24, and 36 h). The cell viability is expressed as OD values. The morphology of both of the ESCC lines was observed under an inverted phase-contrast microscope after the cells were treated for 24 h, and images were obtained. Significant cell shrinkage and a decreased cellular attachment rate were observed in the ICA treatment groups. All of the results are expressed as the mean ± SD; n = 6. aP < 0.05 vs. the control group; bP < 0.05 vs. the 20 μM ICA-treated group; cP < 0.05 vs. the 40 μM ICA-treated group.

Mentions:
To investigate whether ICA displays antitumor activity in EC109, TE1, and HET-1A cells, the CCK-8 assay was used to evaluate the effects of ICA on different cells, and the data are shown in Fig. 1. Treatment of EC109 and TE1 cells for 12, 24, or 36 h with 20, 40, or 80 μM ICA inhibited cell viability in a dose- and time-dependent manner. The 50% inhibitory concentrations (IC50s) of ICA at 12, 24, and 36 h were approximately 106.13 μM, 73.65 μM, and 38.59 μM in EC109 cells and 115.29 μM, 76.77 μM, and 42.21 μM in TE1 cells, respectively. Microscopic images (EC109 in Fig. 1A and TE1 in Fig. 1B) indicated that compared with the control treatment ICA treatment resulted in significant cell shrinkage (based on the bar and cellular gap) and reduced the rate of cellular attachment (based on the cell numbers). However, ICA at the same concentrations did not affect the survival of human primary esophageal epithelial HET-1A cell (Supplementary Fig. 2). The results suggest that icariin has promising anti-ESCC activity with low cytotoxic effect on normal esophageal epithelial cell.

Bottom Line:
ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs.The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment.In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.

ABSTRACTIn this study, we investigated the antitumor activity of icariin (ICA) in human esophageal squamous cell carcinoma (ESCC) in vitro and in vivo and explored the role of endoplasmic reticulum stress (ERS) signaling in this activity. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human EC109 and TE1 ESCCs. Additionally, ICA exhibited strong antitumor activity, as evidenced by reductions in cell migration, adhesion, and intracellular glutathione (GSH) levels and by increases in the EC109 and TE1 cell apoptotic index, Caspase 9 activity, reactive oxygen species (ROS) level, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Furthermore, ICA treatments upregulated the levels of ERS-related molecules (p-PERK, GRP78, ATF4, p-eIF2α, and CHOP) and a pro-apoptotic protein (PUMA) and simultaneously downregulated an anti-apoptotic protein (Bcl2) in the two ESCC cell lines. The downregulation of ERS signaling using eIF2α siRNA desensitized EC109 and TE1 cells to ICA treatment, and the upregulation of ERS signaling using thapsigargin sensitized EC109 and TE1 cells to ICA treatment. In summary, ERS activation may represent a mechanism of action for the anticancer activity of ICA in ESCCs, and the activation of ERS signaling may represent a novel therapeutic intervention for human esophageal cancer.